Human malignancies exhibit elevated levels of homologous recombinational (HR) DNA repair proficiency, and we propose that this common feature of malignancy can be exploited therapeutically. We have supported this central hypothesis by developing drug candidates that specifically inhibit HR and overcome the treatment resistance associated with HR up-regulation in cancer cells. Our renewal application builds on these drug discovery efforts and explores novel applications for compounds that target the central HR protein, RAD51. The major hypotheses and goals of this proposal are as follow: First, we will further optimize RAD51-inhibitory compounds with the goal of overcoming tumor resistance to chemotherapy and radiotherapy. We will develop a novel class of compounds that target a specialized activity of RAD51, which we hypothesize will generate less toxicity than more generalized RAD51 inhibitors. Second, we hypothesize that RAD51-inhibitory compounds will inactivate an HR-related mechanism called alternative lengthening of telomere (ALT), which is required for cell proliferation in some cancer subtypes. Since normal human cells exclusively utilize telomerase instead of ALT to maintain their telomeres, we predict that RAD51 inhibition will force ALT-dependent tumor cells into senescence while exerting little or no normal tissue toxicity. Third, we hypothesize that RAD51-stimulatory compounds can specifically promote death in tumor cells that overexpress RAD51. This concept builds on our observations that high levels of RAD51 overexpression cause the formation of toxic RAD51 protein complexes on undamaged chromatin in cancer cells. We have also shown that RAD51-stimulatory compounds accentuate this potentially toxic feature in susceptible cancer types. All of these hypotheses wil be tested using the same three integrated aims, which incorporate a wide range of drug development methods. The first aim will consist of medicinal chemistry optimization and ADMET testing. Specifically, the structures of our lead RAD51-modulating compounds will be optimized via targeted chemical modifications aimed at improving both activity and pharmacologic properties. In the second aim, we will characterize the activity and specificity of RAD51-modulating compounds, using both purified in vitro biochemical systems and cell-based assays. The third aim will validate the highest priority compounds in animal models. RAD51-inhibitory compounds will be tested in two mouse models to confirm that they: 1) sensitize human tumor xenografts to treatment with replication-disrupting chemotherapeutic drugs and/or radiation, and 2) prevent the ALT-dependent cancer cells from forming lung metastases in mice. RAD51- stimulatory compounds will be tested for the ability to shrink susceptible tumor types, by catalyzing toxic RAD51 protein aggregation on undamaged chromatin. Our ultimate goal is to generate two or three drug candidates that are suitable for extended pharmacologic testing and subsequent testing in clinical trials.